Mark Stadtfeld and his colleagues at the NYU Longone Medical Center has devised a new method for making induced pluripotent stem cells that greatly increases efficiency at which these cells are made.
Induced pluripotent stem cells or iPSCs are made from mature, adult cells by mean of a combination of genetic engineering and cell culture techniques. In short, the expression of four genes is forced in adult cells; Oct4, Sox2, Klf4, and c-Myc or OSKM. The proteins encoded by these four genes cooperatively work to drive a fraction of the cells into an immature state that resembles that of embryonic stem cells. These cells are them grown in cell culture systems that select for those cells that can grow continuously and form colonies of cells derived from progenitor cells. These cell colonies are them repeated isolated a re-cultured until an iPSC line has been established.
Unfortunately, this process is rather inefficient and tedious, since less than one percent or so of the reprogrammed cells actually undergo successful reprogramming. Additionally, it can take several weeks to properly establish an iPSC line. Thus, stem cell scientists have been looking at several different ways to boost the efficiency of this process.
Stadtfeld and his coworkers tried to add compounds to the cultured cells to determine if the culture conditions could actually augment the efficiency of the reprogramming process. “We especially wanted to know if these compounds could be combined to obtain stem cells at high-efficiency,” said Stadtfeld.
The compounds to which Stadtfeld was referring were two cell signaling proteins called Wnt and TFG-beta. Both of these compounds regulate a host of cell growth processes. Stadtfeld wanted to try regulating both of these pathways at the same time, in addition to providing cells with ascorbic acid, which is also known as vitamin C. Even vitamin C is more popularly known as an antioxidant, vitamin C also can remodel chromatin (that tight structure into which cells package their DNA).
When mouse skin fibroblasts were treated with OSKM and a compound that activates Wnt signaling, the efficiency of iPSC derivation increased slightly. The same thing was observed if fibroblasts were treated with OSKM and a compound that inhibits TGF-beta signaling or vitamin C. However, when all three of these compounds were combined, OSKM-engineered fibroblasts were reprogrammed at an efficiency of close to 80 percent. When different cell types were used as the starting cell, such as blood progenitor cells, the efficiency jumped to close to 100 percent; a result that was also observed if liver progenitor cells were used as the starting cell.
Stadtfeld is confident that these dramatic increases in iPSC derivation should improve future studies with iPSCs, since his protocol should make iPSC derivation more predictable. “It’s just a lot easier this way to study the mechanisms that govern reprogramming, as well as detect any undesired features that might develop in iPSCs,” he said.
Vitamin C and the two compounds used to manipulate the Wnt and TGF-β pathways have been widely used in research and have few unknown or hazardous effects. However, OKSM has in some cases caused undesired features in iPSCs, such as increased mutation rates. Stadtfeld believes that by making iPSC induction more rapid and efficient, his new technique might also make the resulting stem cells safer. “Conceivably it reduces the risk of abnormalities by smoothening out the reprogramming process,” Dr. Stadtfeld says. “That’s one of the issues we’re following up.”